Recent epidemiological evidence in humans linking air pollution with increased adiposity and metabolic diseases has garnered considerable interest in the use of mouse models to identify potential underlying biological mechanisms. In this regard, limited studies have shown that exposure to concentrated ambient particulate matter (PM) increases adiposity and insulin resistance in the context of an obesogenic diet. Furthermore, these studies have focused on PM2.5 (aerodynamic diameter S 2.5pm) and been done using cross-sectional study designs with only one exposure period. By comparison, studies involving central nervous system phenotypes have employed nanoscale PM (nPM; aerodynamic diameter 2 200nm), which have been shown to enter the brain where they have functional effects. Although both PM2.5 and nPM reliably induce oxidative stress and inflammation in tissues, nPM have steep near-roadway gradients corresponding to the associations between near-roadway air pollution (NRAP) and obesity in the Children's Health Study (CHS) described in Project 1. Despite these associations, the sequence of metabolic and/or inflammatory changes that lead to obesity are not known. Understanding these pathophysiological mechanisms could thus have important implications for protecting the population from air pollution exposures of greatest current and future concern. To address these critical barriers. Project 3 will carry out comprehensive experiments with the wellestablished C57BL/6 mouse model of obesity and will combine litter reduction at birth (to induce over-nutrition dunng early life) with high fat feeding at the time of weaning. Using a longitudinal study design, mice will be exposed to a novel near-roadway source of nPM (exposed group) or filtered air (control group) dunng prenatal, postnatal, or both pre and postnatal development. Mice will be characterized for obesity-related metabolic, molecular, biochemical, and neurobiological phenotypes at 5 weeks of age (puberty period), 9 weeks of age (late adolescence), and 13 weeks of age (young adulthood). In Specific Aim 1, we will determine body composition (lean tissue mass and whole body fat) by magnetic resonance imaging, assess glucose/insulin metabolism by intraperitoneal glucose tolerance tests (IPGTTs), measure a panel of adipocytokines in plasma, and determine hepatic lipid content. In Specific Aim 2, we will characterize adipose tissue from mice by immunohistochemistry to determine the presence of crown-like structures (CLS), which is indicative of macrophage infiltration and inflammation, and by flow cytometry to quantitate macrophage subtypes (M1/M2). Explant incubation studies will be carried out to determine in wfro production of adipocytokines and real-time PCR will be used to investigate inflammatory and metabolic gene expression in both isolated adipocytes and macrophages. In Specific Aim 3, we will determine whether the effects of nPM exposure on obesity are mediated through neurobiological pathways in the hypothalamus that control metabolic regulation. Immunohistochemical techniques and morphometric analyses will be used to characterize the organization of hypothalamic neural projections involved in feeding regulation. Expression of metabolically-relevant neuropeptide genes will also be investigated in a nucleus-specific manner by real-time PCR and food intake will be assessed to determine whether nPM exposure results in altered feeding behavior. Taken together, the proposed studies offer several levels of innovation: (1) consistent with the Center focus on NRAP, we will use nPM collected near a major traffic corridor and which we have previously shown to have demonstrable biochemical and molecular effects in vitro and in vivo; (2) the nPM reflects the nearroadway gradient in the biologically relevant nanoscale size fraction that is ennched in elemental carbon and metals of vehicular source; (3) the novel collection and exposure procedure will preserve the size distnbution of the original aerosol and the known potential of such nPM to translocate into the systemic circulation and into organs, including the brain; (4) the mouse model of obesity reflects the natural life course of obesity in humans by coupling over-nutrition in early life with high fat feeding at weaning; and 5) exposing animals to nPM during three developmental stages will identify critical window(s) of susceptibility. As a result. Project 3 is highly integrated with the other projects of this Center and will complement the human studies by elucidating the pathophysiological mechanisms that underlie the effects of NRAP on obesity and metabolic dysregulation. The physiological phenotypes that we will obtain in mice, such as whole body composition, glucose tolerance, and hepatic fat deposition, will be comparable to those obtained by Project 1, which will study CHS participants selected from the extremes of lifetime exposure to air pollution. The murine phenotypes obtained at the levels of adipose tissue, including histology, adipocytokine release, and cell-specific gene expression, will similarty be equivalent to those being obtained from CHS subjects as proposed in Project 2. Thus, the experiments proposed in Project 3 will yield meaningful insight into the temporal sequence and directionality of the effects of nPM on three important aspects of obesity in mice and may provide causal information that could guide informed analyses in Projects 1 and 2, as analogous obesity-related parameters are collected in humans.